Matrix-induced Myogenesis & Pharmaco-Screens of MSCs Mesenchymal Stem Cells (MSC) are capable of differentiating into many tissue lineages, including muscle, leading to the belief that such cells contribute to homeostatic repair processes. In many diseases, including muscular dystrophies, tissue matrix is abnormal and progenitor contributions to repair are inadequate. Our goal is to develop tissue-mimetic cell culture models of normal and diseased myogenesis, focused on the influence of normal and abnormal matrix elasticity, in order to assess known drugs (eg. Prednisolone) and ultimately screen an NIH library for drug candidates that steer matrix-coupled myogenesis of stem cells. As a specific disease, we also focus on nucleo-dystrophies because of relevance to muscle and also for deeper insight into differentiation mechanisms. The nuclear lamina interacts with chromatin and through linker proteins that span the envelope the lamina also interacts with the cytoskeleton. We have shown recently that the cytoskeleton is strongly influenced by matrix elasticity in acto-myosin striation of skeletal muscle cells [Engler J. Cell Biology 2004] as well as in multi-lineage differentiation of MSCs [Engler Cell 2006]. Adhesion to matrix physically couples to myosin-based contractility in these cells and most other tissue cells, and so a linkage is formed from the matrix through the cytoskeleton and into the normal nucleus. This continuous linkage from nucleus~cytoskeleton~matrix makes MATRIX ELASTICITY A CRITICAL BIOLOGICAL INPUT. This R21's "develop and explore" objectives will therefore (1) adapt our elastic matrices to both low & high throughput screening formats and eventually screen for novel myo-differentiation inducers on normal human MSC on abnormal matrix (that mechanically mimics fibrosis), and will (2) develop a myogenic disease model both for the screen and to test the hypothesis that nuclear defects strongly influence myogenic differentiation and nuclear plasticity. In just serum media, we will use matrix elasticity to control myogenic differentiation of normal MSC and also, importantly, to mimic rigid and fibrotic tissues typical in disease. The same matrix system allows comparisons to osteogenesis on rigid matrices and neurogenesis on soft, brain- mimetic matrices. Soluble induction factors (eg. glucocorticoids) that enhance or inhibit the lineages will be screened and studied for safety & efficacy with a particular goal to promote myogenesis. We will also engineer human MSC to express known dystrophy causing mutants such as those in Lamin-A/C, while knocking down endogenous protein to minimize over-expression artifacts. With select differentiated states and nuclear mutants treated with lead drugs from the screen, insights into protein folding and association state of nuclear components such as lamins within the intact cell will be probed by a novel proteomic-scale Cys Shotgun labeling strategy that exploits quantitative Mass Spectrometry methods [Johnson 2007]. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: We propose to develop an elastic matrix culture format for multi-well screens of soluble factors that influence myogensis of adult-derived mesenchymal stem cells. The various elastic matrices mimic the influential variation of tissue mechanical properties in normal and diseased states. We will apply the screen to normal human stem cells on abnormal matrices (that mechanically mimic fibrosis), and ultimately to re- engineered cells expressing known muscular dystrophy-causing mutants such as those in Lamin-A/C. A goal will be to understand mechanistic blocks to differentiation and also to screen known compounds and eventually libraries that over-ride matrix signals and promote differentiation in diseased cells. [unreadable] [unreadable] [unreadable]